Exposure of animal cells to interferon protects them against subsequent viral infections. Interferon activates cellular defence mechanisms, the molecular basis of which is still largely unknown. Recently, two enzymatic activities have been shown to increase in interferon-treated cells: a protein kinase, which phosphorylates initiation factor eIF-2 and causes inhibition of protein synthesis, and an enzyme which synthesizes 2'5' oligo (A) from ATP. Both enzymes are activated by double-stranded RNA (dsRNA). The 215'oligo (A) is in turn the activator of an endonuclease, which is present constitutively in the cells so far examined. The objective of the proposed research is to understand how an increase in these enzymatic activities interferes with virus replication. In particular, we would like to understand the precise mechanism of discrimination, the process by which interferon-treated cells distinguish from cellular templates. Human and mouse tissue culture lines will be used for these studies. Fibroblast interferon will be used to treat cells and the induction of protein kinase and 2'5' oligo (A) polymerase upon treatment with interferon will be correlated with its antiviral effects. Control and interferon-treated cells will be infected with encephalomyocarditis or vesicular stomatitis virus in these studies. In a parallel study the activation of protein kinase and 2'5' oligo (A) polymerase with synthetic dsRNAs of different size and base composition will be studied to establish whether these activators share molecular features common with interferon inducers. Assays for both protein kinase and 2'5' oligo (A) polymerase, which provide a rapid and quantitative determination of activity in cell extracts have been developed for this purpose. Studies in intact animals infected with viruses or treated with interferon will establish whether these enzymes are increased in vivo; an explanation for the surprising finding that a large increase in 2'5 oligo (A) polymerase is found in serum of virus-infected animals will be sought.